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. 2016 Jul 21;113(34):9498–9503. doi: 10.1073/pnas.1604371113

Fig. 3.

Fig. 3.

Ig5FN1 is polysialylated when expressed in glycoengineered N. benthamiana. (A) Expression of Ig5FN1 was monitored by SDS/PAGE and Coomassie Brilliant Blue staining of intercellular fluid (IF) derived from ΔXTFT and ΔXTFTSia. A strong band at ∼35–40 kDa corresponds to the glycosylated Ig5FN1 polypeptide. Protein bands ≤35 kDa are associated with agrobacterial infection. IF from infiltrated leaves with Agrobacteria carrying an “empty” plasmid served as a negative control (C). Apparent protein molecular masses are indicated in kDa (M). Glycan analysis of IF-derived Ig5FN1 was performed by LC-ESI-MS of tryptic glycopeptides. The spectra for ASN5 (R/D38GQLLPSSNYSNIK51) are shown. (B) Western blot analysis using mAb735 of IF extracted from ΔXTFTSia infiltrated with (lane 1) Ig5FN1, (lane 2) Ig5FN1 coexpressed with human core αI,6-fucosyltransferase (FUT8), (lane 3) Ig5FN1 coexpressed with both ST8Sia-II and -IV (polySTs), and (lane 4) Ig5FN1 coexpressed with FUT8 and polySTs. As a control sample, (lane C) polySTs were coexpressed with FUT8 in the absence of Ig5FN1. Sample (4) was incubated without (−) and with (+) EndoN. (C) To determine the degree of polymerization (DP), purified polysialylated Ig5FN1 was labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) and separated by HPLC. The degree of polymerization is given for selected peaks.